The cerebellum plays an important role in motor coordination, such as walking, in which plural muscles are involved. Damage of cerebellum results in unsuccessful regulation of motor coordination and failed smooth movement. Motor commands from the cerebral cortex are transmitted through the brain stem to the spinal cord and muscles, while these commands are also transmitted from the brain stem (pons) via mossy fibers to the cerebellar cortex. The signals transmitted via mossy fibers are input to granule cells which are cerebellar cortex neurons. Then, the granule cells transmit the signals to Purkinje cells via their axons, parallel fibers.
One Purkinje cell forms 100,000 or more synapses with the parallel fibers. The Purkinje cells integrate input information therefrom and output the information to the cerebellar nuclei located in the deep region of the cerebellum. The Purkinje cells, the sole output neurons from the cerebellar cortex, play a very important role in the cerebellum. However, they are fragile and are easily damaged due to cerebellar hemorrhage, injury, cerebellar tumor, or inherited neurodegenerative disease.
With the developments in genomics in recent years, the human and mouse complete genome sequences have been determined. Life science research is now headed for the elucidation of roles of identified genes. An approach which involves introducing a wild-type or mutant gene to neurons and examining its influence is very effective from the viewpoint of research in the neuroscience field. Such gene transfer to neurons is also getting attention from the viewpoint of clinical application as gene therapies.
However, the gene transfer to neurons is difficult, because they are nondividing cells. Among others, gene transfer to Purkinje cells is exceedingly difficult. Prior to 2000, successful cases of efficient gene transfer to Purkinje cells were not reported. Recent progress in the development of viral vectors has permitted efficient gene transfer to Purkinje cells. Nevertheless, only 4 articles have been reported so far (Non-Patent Documents 1 to 4: Agudo M., Trejo J. L., Lim F., Avila J., Torres-Aleman I., Diaz-Nido J. & Wandosell F. (2002) Highly efficient and specific gene transfer to Purkinje cells in vivo using a herpes simplex virus I amplicon. Hum. Gene Ther., 13, 665-674; Alisky J. M., Hughes S. M., Sauter S. L., Jolly D., Dubensky T. W. Jr., Staber P. D., Chiorini J. A. & Davidson B. L. (2000) Transduction of murine cerebellar neurons with recombinant FIV and AAV5 vectors. Neuroreport, 11, 2669-2673; Kaemmerer W. F., Reddy R. G., Warlick C. A., Hartung S. D., McIvor R. S. & Low W. C. (2000) In vivo transduction of cerebellar Purkinje cells using adeno-associated virus vectors. Mol. Ther., 2, 446-457; and Xia H., Mao Q., Eliason S. L., Harper S. Q., Martins I. H., Orr H. T., Paulson H. L., Yang L., Kotin R. M. & Davidson B. L. (2004) RNAi suppresses polyglutamine-induced neurodegeneration in a model of spinocerebellar ataxia. Nat. Med., 10, 816-820).
Efficient gene transfer to Purkinje cells requires the use of vectors based on viruses, such as a lentivirus or adeno-associated virus, which have high affinity for neurons. For information, an adenovirus, a herpesvirus, an adeno-associated virus, and a feline immunodeficiency virus-derived lentivirus have been used in the gene transfer to Purkinje cells in the previous reports.
A wild-type lentivirus infects only lymphocytes having CD4 receptors. Therefore, lentiviral vectors currently used have their envelope proteins substituted by a vesicular stomatitis virus glycoprotein (VSV-G) such that they also infect neurons. This VSV-G has the ability to bind to phospholipid (phosphatidylserine) serving as a component of cell membranes and therefore nonspecifically infects non-neuronal cells such as glial cells and vascular endothelial cells. Moreover, in the cerebellum, the lentivirus also infects stellate cells, basket cells, Golgi cells, and Bergmann glia, in addition to Purkinje cells.
As described above, an adeno-associated viral or lentiviral vector has the ability to introduce a foreign gene to neurons and express the gene. However, these viral vectors have a limitation of the size of a gene which can be expressed, due to the problems of their packaging abilities. The size of a gene which can be expressed is allegedly 4 kb and 8 kb, including promoter regions, for adeno-associated viral and lentiviral vectors, respectively. However, this is a theoretical value. Lentiviral vector plasmids are variously modified for increasing safety. Therefore, gene size to be expressed is often limited to around 5 kb, though it differs depending on vector plasmids used.
On the other hand, an L7 promoter, which is active specifically in Purkinje cells, has been utilized in the production of transgenic mice expressing a foreign gene only in Purkinje cells. However, the L7 promoter is as large as approximately 3 kb in full length and has a large limitation of the size of a foreign gene to be expressed, when inserted in an adeno-associated viral or lentiviral vector for use. Moreover, its promoter activity is much lower than that of a nonspecific promoter such as a CMV or MSCV promoter.
Efficient and specific gene transfer to cerebellar Purkinje cells has been achieved only by a method which involves producing transgenic mice. However, specific and highly efficient gene transfer to Purkinje cells using viral vectors will permit various studies never before achieved and largely contribute to developments in studies focused on the cerebellum. Moreover, such gene transfer is also expected to be applied to gene therapies for Purkinje cell-affecting diseases such as spinocerebellar ataxia.
Moreover, infections including bovine spongiform encephalopathy have presented large problems in recent years, when viral vectors are used in humans for gene therapies. This is because current protocols require adding bovine serum to a medium during virus generation. Thus, if a viral vector that achieves highly efficient gene transfer to Purkinje cells can be generated without the use of serum, its clinical application is expected to be more expanded.